A full-length cDNA clone encoding horse Mhc class II DQA antigen has been isolated and sequenced. A lymphocyte-derived cDNA library was constructed from an Eqca class I A2 homozygous stallion and cloned into pcDNA I vector (Barbis et al. 1994). The library was screened according to Twomey and Krawetz (1990). The probe for screening was obtained by amplifying the second exon of the DQA gene from a 10 -4 dilution of the cDNA library itself. For this purpose primers were designed upon conserved regions among mammalian species. The sequences were: 5'-GTGATGAGCCCCTGTGGAGGT-T and 5'-CACTGTGACCTCAGGAACCT-3'. First the pcDNA-I clones were sequenced with vector primers (T7, SP6). At the end of the sequences, new primers were designed and used in polymerase chain reaction (Fig. 1). The fragments were cloned into pT7Blue T-Vector system (Novagen, WI) and both strands were sequenced with vector primers (T7, U19).
The Eqca-DQA cDNA clone was found to be 1115 basepairs in length and the protein it most probably encodes consists of 232 amino acids from the first residue of the cd domain (Fig. 1). The signal peptide consists of 23 residues. The length of the predicted horse DQA peptide chain corresponded with those found in other mammalian species. The signal peptide and the mature peptide showed high sequence similarity to DQA sequences from other species (85% with human [GenBank AC M33906], 83% with sheep [GenBank AC M93431], 84% with pig [Gen-Bank AC M29938), 80% with mouse H2-IA [GenBank AC M21931], and 82% with cattle [GenBank AC M90306]). Only 51% sequence similarity was found with the horse DRA locus, as an example of higher sequence similarity